Summary Document
This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.
http://www.cyto.purdue.edu/hmarchiv/index.htm
Thank you for your replies. Here are the answers I received concerning
'electrical noise in FACS Aria'.
When I off the agitation this extra peak disappeared. (My aria doesn't
have temperature control unit.)
=============================================================
QUESTION:
When I run my negative control sample in three year old Aria I found
the peak is moving left and right, then right shoulder population is
produced. These things happened during the collection. When I changed
compensation number this peak is not moving. So, is this electrical
noise or software problem? I am using DIVA ver 4.0.1.2.
REPLIES:
-------------------------------------------------
1. This noise or extra peak in your population maybe due to agitation
and temperature control , try turning this two off.
Also try to observed when the agitation of sample is ongoing you will
notice the "extra peak" coming out during the acquisition . We have the
same problem with our Aria for a year now, BD people know about this
problem and working for a solution. Mr.J Ilas
-------------------------------------------------
2. I saw your posting on the Purdue message board, is your sample
agitator on when this happens? I sometimes see a shift in population
that coincides with the agitator turning on and off, Field Service is
going to be here on Thursday to replace a few parts to try and
troubleshoot it. I also sometimes see it with the temperature control
turned on. I'll let you know what happens after our repair. Mike Waring
-------------------------------------------------
3. We see something similar on our two year old FACS Aria. When we cool
the sample and use the sample agitation we see a big change in signals.
The field engineer installed an extra power supply and changed the other
which solved the problem half. We can use the cooling without problem
but have still the problem with the sample agitation. Our opinion is
that there is no relation whit the software (we use version 4.1.2
"20050606") Frank van Diepen
-------------------------------------------------
4. The standard deviation of electronic noise is probably somewhere near
12 - however, your green detector gain is so low (appears < 100) that a
lot of what is there is "noise" anyway. Using the 5 decade range is
tricky because it can give you a false sense of how well populations are
being measured, and I would recommend you use the 4 decade range where
the minimum value is 26 instead of 3. Without seeing where the positives
go it is difficult to say for sure, but it looks like you might benefit
from boosting the detector gain. If the GFP is leaking and contributing
to the signal from the sample buffer, then baseline may change over time
in some samples. There does not appear to be any correlation to
compensation, so you would not expect compensation to do anything. This
is likely a combination of sample behavior (background GFP or not) with
too low a gain setting, and has nothing to do with software. As a
general rule, you might want to put the median of unstained at minimally
about 10 times noise, which would be probably between 80 and 160 on that
system, and rule out effects from the sample such as leaked out GFP in
the buffer. Most cells have significant auto fluorescence in the "FITC"
detector, and getting them well on-scale should not be a problem. Joe
-------------------------------------------------
5. This sounds like a fluidics problem to me. If the peaks are drifting
during acquisition you should be able to easily see this on a dot plot
time vs fitc. You should also catch this on the QC- If this is the
case, I think it is either a dirty flow cell or your loosing pressure
during the run. For me, this often happens when the seal around the
agitation motor begins to go- mine usually do not last 6months.
Although, there a host of other possibilities- sounds like a call to
BDIS. Jim.
-------------------------------------------------
6. The problem looks like auto fluorescence to me. Noise usually occurs
to the far left. do you see this with beads or with cells only? Mary
=============================================================
